Non-Human Primate Cytokine Magnetic Bead Panel 96 Well ... · Anti- IL-2 Bead 33 ˜ PRIL2-MAG ˜...

Non-Human Primate Cytokine Magnetic Bead Panel 96 Well Plate Assay Cat. # PRCYTOMAG-40K Cat. # PCYTMG-40K-PX23

MILLIPLEX® MAP

NON-HUMAN PRIMATE CYTOKINE MAGNETIC BEAD PANEL KIT 96 Well Plate Assay

# PRCYTOMAG-40K

# PCYTMG-40K-PX23

TABLE OF CONTENTS PAGE

Introduction 2

Principle 4

Storage Conditions Upon Receipt 4

Reagents Supplied 5

Materials Required But Not Provided 7

Safety Precautions 7

Technical Guidelines 8

Sample Collection And Storage 9

Preparation of Reagents for Immunoassay 11

Immunoassay Procedure 14

Plate Washing 16

Equipment Settings 16

Quality Controls 18

Assay Characteristics 19

Troubleshooting Guide 23

Replacement Reagents 26

Ordering Information 27

Well Map 28

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

By purchasing this product, which contains fluorescently labeled microsphere beads authorized by Luminex Corporation (“Luminex”), you, the customer, acquire the right under Luminex’s

patent rights, if any, to use this product or any portion of this product, including without limitation the microsphere beads contained herein, only with Luminex’s laser based fluorescent analytical

test instrumentation marketed under the name of Luminex 100TM IS, 200TM, HTS, FLEXMAP 3DTM , MAGPIX®.

PRCYTOMAG-40K Rev. 17-JAN-2012 PAGE 2 EMD MILLIPORE

Introduction “Cytokine” is a general term used for a diverse group of soluble proteins and peptides which act as regulators under both normal and pathological conditions to modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Cytokines differ from hormones in that they act on a wider spectrum of target cells and also they are not produced by specialized cells which are organized in specialized glands. This group of proteins includes lymphokines, interferons, colony stimulating factors and chemokines. Cytokine and chemokine research plays a significant role in achieving a deeper understanding of disease states such as allergic reactions, IBD, sepsis, and cancer. Therefore, the MILLIPLEX MAP Non-Human Primate Cytokine Panel enables you to focus on the therapeutic potential of cytokines as well as the modulation of cytokine expression. Coupled with the Luminex xMAP® platform, you receive the advantage of ideal speed and sensitivity, allowing quantitative multiplex detection of dozens of analytes simultaneously which can dramatically improve productivity. Using Luminex xMAP technology, EMD Millipore has developed the MILLIPLEX MAP Non-Human Primate Cytokine Multiplex Panel. Magnetic Beads can make the process of automation and high throughput screening easier with features such as walk-away washing. Advantages even outside automation include:

More flexible plate and plate washer options. Improved performance with turbid serum/plasma samples. Assay results equivalent to non-magnetic beads. Automated washing eliminates technical obstacles (e.g., clogging of wells that

contain viscous samples) which may result during vacuum manifold/manual washing.

EMD Millipore’s MILLIPLEX MAP Non-Human Primate Cytokine panel is the most versatile system available for Cytokine research.

MILLIPLEX MAP offers you the ability to: o Select a 23-plex premixed kit or o Choose any combination of analytes from our panel of 23 analytes to

design a custom kit that better meets your needs. A convenient “all-in-one” box format gives you the assurance that you will have

all the necessary reagents you need to run your assay. EMD Millipore’s MILLIPLEX MAP Non-Human Primate Cytokine panel kit is to be used for the

simultaneous quantification of G-CSF, GM-CSF, IFNγ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23(p40), IL-13, IL-15, IL-17A, MCP-1, MIP-1β, MIP-1α, sD40L, TGF-α, TNF-α, VEGF, and IL-18. This kit may be used for the analysis of all or any combination of the above analytes in tissue/cell lysate and culture supernatant samples and serum or plasma samples

This kit is for research purposes only.

Please read entire protocol before use.


It is important to use same assay incubation conditions throughout your study. Alternative Names of Cytokines/Chemokines

Cytokine Name Alternative Name

G-CSF Granulocyte colony stimulating factor DF, GM-DF, MGI-2, NAP-IF, Pluriopoietin, PCSF, 5637-derived factor, Pluripoietin-beta

GM-CSF Granulocyte-macrophage colony stimulating factor

CSF-alpha, CSF-beta, CSF-2, FDCP1 growth factor, GM-CSA, HCSF, KM102-BPA, KTGF, LBGF, NIF-T, Pluripoietin- alpha, TPO, TSF, burst promoting activity (BPA)

IFN Gamma Interferon Antigen induced Interferon, immune interferon, type 2 interferon, T-interferon, Mitogen induced interferon, pH2-labile interferon

IL-1β Interleukin 1 Beta Catabolin, H1, IFN-beta inducing factor, Interleukin -beta, OAF,

IL-1ra IL-1 receptor antagonist IL1RN; IL1RA; ICIL-1RA; IL-1ra3; IL1F3; IRAP; MGC10430

IL-2 Interleukin 2 BF, Costimulator, LMF, LCM factor, LPF, MAF-C1, PFC-EA, SCIF, TCGF, TCPA, TDF, T-LPF, TMF, TSF

IL-4 Interleukin 4 IaIF , BSF-1, BCDF-gamma, BCGF-gamma , BCGF-1, BSF-1, BSF-p1 , EL4-BCGF, IgE-EF, IgE switch factor, IgG1-enhancing factor, IgG1-induction factor, LMW-BCGF, MCGF-2 , TCGF-2

IL-5 Interleukin 5 B151-TRF , BCDF-mu , BCGF-2, DL-BCGF, CFU-Eo GSF, EDF, Eo-CSF, Eo-DF, IgA-EF, TRF-1

IL-6 Interleukin 6 26 kDa protein, BSF-2 ,BSF-p2 , CSF-309 , DIF, FDGI , HGI, HSF, HSF-1,IFN-beta-2, ILHP1 , MGI-2A , Myeloma GF , Natural killer cell activity-augmenting factor , WI-26-VA4 factor , CPA

IL-8 Interleukin 8 3-10C; AMCF-I; CXCL8; GCP-1; GCP1; K60; LECT; LUCT; LYNAP; MDNCF; MONAP; NAF; NAP-1; NAP1; SCYB8; TSG-1; b-ENAP

IL-10 Interleukin 10 B-TCGF, CSIF, TGIF

IL-12/23(p40) Interleukin 12, heterodimeric glycoprotein CLMF, NKSF, TSF

IL-13 Interleukin 13 NC30

IL-15 Interleukin 15 IL-T

IL-17A Interleukin 17 IL-17A

MCP-1 Monocyte chemoattractant protein-1 SCYA2, CCL2, GDCF, HC11, LDCF, MCAF, MCP, SMC-CF, TDCF,TSG-8

MIP-1α Macrophage inflammatory protein-1-alpha CCL3

MIP-1β Macrophage inflammatory protein-1-beta CCL4, ACT-2, LAG-1

sCD40L soluble CD40 ligand TRAP, CD154

TGFα Transforming growth factor alpha ETGF, TFGA

TNFα Tumor necrosis factor-alpha Cachectin, cytotoxic factor (CF), CTX, Hemorrhagic factor, Macrophage-derived cytotoxic factor, macrophage cytotoxic factor (MCF), MCT, TNFSF2

VEGF Vascular endothelial growth factor Mouse sarcoma 180-derived growth factor, Vasculotropin.(VAS), Vascular endothelial cell proliferation factor , VPF, GD-VEGF, VEGF-A

IL-18 Interleukin 18 IGIF; IL-1g; IL1F4; MGC12320


PRINCIPLE

MILLIPLEX MAP are based on the Luminex® xMAP® technology — one of the fastest growing and most respected multiplex technologies offering applications throughout the life-sciences and capable of performing a variety of bioassays including immunoassays on the surface of fluorescent-coded magnetic beads known as MagPlexTM-C microspheres.

Luminex® uses proprietary techniques to internally color-code microspheres with two fluorescent dyes. Through precise concentrations of these dyes, 100 distinctly colored bead sets can be created, each of which is coated with a specific capture antibody.

After an analyte from a test sample is captured by the bead, a biotinylated detection antibody is introduced.

The reaction mixture is then incubated with Streptavidin-PE conjugate, the reporter molecule, to complete the reaction on the surface of each microsphere.

The microspheres are allowed to pass rapidly through a laser which excites the internal dyes marking the microsphere set. A second laser excites PE, the fluorescent dye on the reporter molecule.

Finally, high-speed digital-signal processors identify each individual microsphere and quantify the result of its bioassay based on fluorescent reporter signals.

The capability of adding multiple conjugated beads to each sample results in the ability to obtain multiple results from each sample. Open-architecture xMAP® technology enables multiplexing of many types of bioassays reducing time, labor and costs over traditional methods. STORAGE CONDITIONS UPON RECEIPT

Recommended storage for kit components is 2 - 8°C.

For long-term storage, freeze reconstituted standards and controls at -20°C. Avoid multiple (>2) freeze/ thaw cycles.

DO NOT FREEZE Antibody-Immobilized Beads, Detection Antibody, and Streptavidin-Phycoerythrin.


REAGENTS SUPPLIED Note: Store all reagents at 2 – 8°C

REAGENTS SUPPLIED CATALOG NUMBER VOLUME QUANTITY Non-Human Primate Cytokine Standard MXPR8040 lyophilized 1 vial

Non-Human Primate Cytokine Quality Controls 1 and 2

MXPR6040 lyophilized 2 vials

Serum Matrix (for serum/plasma samples only)

MXPRSM lyophilized 1 vial

Set of one 96-Well Plate with 2 sealers ----------- ----------- 1 plates 2 sealers

Assay Buffer Note: Contains 0.05% Proclin

L-AB 30 mL 1 bottle

10X Wash Buffer Note: Contains 0.05% Proclin

L-WB 30 mL 2 bottle

Non-Human Primate Cytokine Detection Antibodies

MXPR1040-2 3.2 mL 1 bottle

Streptavidin-Phycoerythrin

L-SAPE4

3.2 mL 1 bottle

Bead Diluent (not provided with premixed panel)

LBD 3.5 mL 1 bottle

Mixing Bottle (not provided with premixed panel)

------------ -------------- 1 bottle

Non-Human Primate Cytokine Antibody-Immobilized Premixed Beads: Premixed 23-plex Beads PRCYPMX23-MAG 3.5 mL 1 bottle

Included Non-Human Primate Cytokine Antibody-Immobilized Beads are dependent on customizable selection of analytes within the panel (see following table page 7).


Non-Human Primate Cytokine Antibody-Immobilized Magnetic Beads:

Bead/Analyte Name

Luminex magnetic

Bead Region

Customizable 23 Analytes (50X Concentration, 90 µL) Available Cat. #

23-Plex Premixed

Beads

Anti- G-CSF Bead 18 � HGCSF-MAG � Anti- GM-CSF Bead 14 � PRGMCSF-MAG �

Anti- IFN Bead 20 � PRIFNG-MAG � Anti- IL-1β Bead 46 � HCYIL1B-MAG � Anti- IL-1ra Bead 42 � HIL1RA-MAG � Anti- IL-2 Bead 33 � PRIL2-MAG � Anti- IL-4 Bead 53 � HIL4-MAG � Anti- IL-5 Bead 55 � HIL5-MAG � Anti- IL-6 Bead 57 � HCYIL6-MAG � Anti- IL-8 Bead 63 � HCYIL8-MAG �

Anti- IL-10 Bead 35 � PRIL10-MAG � Anti- IL-12/23(p40) Bead 74 � PRIL12P40-MAG �

Anti- IL-13 Bead 44 � PRIL13-MAG � Anti- IL-15 Bead 37 � HIL15-MAG �

Anti- IL-17A Bead 39 � HIL17-MAG � Anti-IL-18 Bead 78 � PRIL18-MAG �

Anti- MCP-1 Bead 67 � HCYMCP1-MAG � Anti- MIP-1β Bead 73 � HMIP1B-MAG � Anti- MIP-1α Bead 65 � PRMIP1A-MAG � Anti- sCD40L Bead 38 � HCD40L-MAG �

Anti-TGFα Bead 15 � HCYTGFA-MAG � Anti- TNFα Bead 72 � PRTNFA-MAG � Anti- VEGF Bead 76 � PRVEGF-MAG �


MATERIALS REQUIRED BUT NOT PROVIDED

Reagents

1. Luminex Sheath Fluid (Luminex Catalogue #40-50000) or Luminex Drive Fluid (Luminex Catalogue # MPXDF-4PK)

Instrumentation / Materials 1. Adjustable Pipettes with Tips capable of delivering 25 L to 1000 L 2. Multichannel Pipettes capable of delivering 5 L to 50 L or 25 L to 200 L 3. Reagent Reservoirs 4. Polypropylene Microfuge Tubes 5. Rubber Bands 6. Aluminum Foil 7. Absorbent Pads 8. Laboratory Vortex Mixer 9. Sonicator (Branson Ultrasonic Cleaner Model #B200 or equivalent) 10. Titer Plate Shaker (Lab-Line Instruments Model #4625 or equivalent) 11. Luminex 200™, HTS, FLEXMAP 3D™, or MAGPIX® with xPONENT software

by Luminex Corporation 12. Automatic Plate washer for magnetic beads (Bio-Tek ELx405, EMD Millipore

catalog #40-015 or equivalent) or Hand held Magnetic Separation Block (EMD Millipore catalog # 40-285 or equivalent)

Note: If a plate washer or hand held magnetic separation block for magnetic beads is not available, one can use a microtiter filter plate (EMD Millipore Catalog #MX-PLATE) to run the assay using a Vacuum Filtration Unit (EMD Millipore Vacuum Manifold Catalog #MSVMHTS00 or equivalent with EMD Millipore Vacuum Pump Catalog #WP6111560 or equivalent).

SAFETY PRECAUTIONS

All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.

Sodium azide or Proclin has been added to some reagents as a preservative. Although the concentrations are low, sodium azide and Proclin may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build up.


TECHNICAL GUIDELINES

To obtain reliable and reproducible results, the operator should carefully read this entire manual and fully understand all aspects of each assay step before running the

assay. The following notes should be reviewed and understood before the assay is set up.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Do not use beyond the expiration date on the label.

Do not mix or substitute reagents with those from other lots or sources.

The Antibody-Immobilized Beads are light sensitive and must be protected from light at all times. Cover the assay plate containing the beads with opaque plate lid or aluminum foil during all incubation steps.

It is important to allow all reagents to warm to room temperature (20-25°C) before use in the assay.

Incomplete washing can adversely affect the assay outcome. All washing must be performed with the Wash Buffer provided.

The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the standard stock which may be stored at -20°C for 1 month and at -80°C for greater than one month.

If samples fall outside the dynamic range of the assay, further dilute the samples with the appropriate diluent and repeat the assay.

Any unused mixed Antibody-Immobilized Beads may be stored in the Mixing Bottle at 2-8°C for up to one month.

During the preparation of the standard curve, make certain to mix the higher concentration well before making the next dilution. Use a new tip with each dilution.

The plate should be read immediately after the assay is finished. If, however, the plate cannot be read immediately, seal the plate, cover with aluminum foil or an opaque lid, and store the plate at 2-8°C for up to 24 hours. Prior to reading, agitate the plate on the plate shaker at room temperature for 10 minutes. Delay in reading a plate may result in decreased sensitivity for some analytes.

The titer plate shaker should be set at a speed to provide maximum orbital mixing without splashing of liquid outside the wells. For the recommended plate shaker, this would be a setting of 5-7 which is approximately 500-800 rpm.

Ensure that the needle probe is clean. This may be achieved by sonication and/or alcohol flushes.


TECHNICAL GUIDELINES (continued

When reading the assay on Luminex 200™, adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 4 alignment discs. When reading the assay on FLEXMAP 3D™, adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 1 alignment disc. When reading the assay on MAGPIX, adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 2 alignment disc.

For cell culture supernatants or tissue extraction, use the culture or extraction medium as the matrix solution in background, standard curve and control wells. If samples are diluted in assay buffer, use the assay buffer as matrix.

For serum/plasma samples that require further dilution use the Serum Matrix provided in the kit for further dilution.

For cell/tissue homogenate, the final cell or tissue homogenate should be prepared in a buffer that has a neutral pH, contains minimal detergents or strong denaturing detergents, and has an ionic strength close to physiological concentration. Avoid debris, lipids, and cell/tissue aggregates. Centrifuge samples before use.

Vortex all reagents well before adding to plate.

SAMPLE COLLECTION AND STORAGE

A. Preparation of Serum Samples: Allow the blood to clot for at least 30 minutes before centrifugation for

10 minutes at 1000xg. Remove serum and assay immediately or aliquot and store samples at -20°C.

Avoid multiple (>2) freeze/thaw cycles. When using frozen samples, it is recommended to thaw the samples

completely, mix well by vortexing and centrifuge prior to use in the assay to remove particulates.

Customers should determine the optimal dilution for their samples. Generally, serum or plasma samples from normal subjects do not need dilution and can be tested directly. However, for those samples that are out of standard range, serum or plasma samples should be diluted using the reconstituted Serum Matrix as the sample diluent.

B. Preparation of Plasma Samples:

Plasma collection using EDTA as an anti-coagulant is recommended. Centrifuge for 10 minutes at 1000xg within 30 minutes of blood collection. Remove plasma and assay immediately or aliquot and store samples at -20°C.

Avoid multiple (>2) freeze/thaw cycles.


SAMPLE COLLECTION AND STORAGE (continued)

When using frozen samples, it is recommended to thaw the samples completely, mix well by vortexing and centrifuge prior to use in the assay to remove particulates.

Customers should determine the optimal dilution for their samples. Generally, serum or plasma samples from normal subjects do not need dilution and can be tested directly. However, for those samples that are out of standard range, serum or plasma samples should be diluted using the reconstituted Serum Matrix as the sample diluent.

C. Preparation of Tissue Culture Supernatant:

Centrifuge the sample to remove debris and assay immediately or aliquot and store samples at -20°C.

Avoid multiple (>2) freeze/thaw cycles. Tissue culture supernatant may require a dilution with an appropriate

control medium prior to assay. Tissue/cell extracts should be done in neutral buffers containing reagents and conditions that do not interfere with assay performance. Excess concentrations of detergent, salt, denaturants, high or low pH, etc. will negatively affect the assay. Organic solvents should be avoided. The tissue/cell extract samples should be free of particles such as cells or tissue debris.

NOTE:

A maximum of 25 μL per well of serum or plasma can be used. Tissue culture or other media may also be used.

All samples must be stored in polypropylene tubes. DO NOT STORE SAMPLES IN GLASS.

Avoid debris, lipids and cells when using samples with gross hemolysis or lipemia.

Care must be taken when using heparin as an anticoagulant since an excess of heparin will provide falsely high values. Use no more than 10 IU heparin per mL of blood collected.


PREPARATION OF REAGENTS FOR IMMUNOASSAY

A. Preparation of Antibody-Immobilized Beads

If premixed beads are used, sonicate the premixed bead vial 30 seconds and then vortex for 1 minute before use. (Note: Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit.)

For individual vials of beads, sonicate each antibody-bead vial for 30 seconds; vortex for 1 minute. Add 60 µL from each antibody bead vial to the Mixing Bottle and bring final volume to 3.0 mL with Bead Diluent. Vortex the mixed beads well. Unused portions may be stored at 2-8C for up to one month. (Note: Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit.)

Example 1: When using 10 cytokine antibody-immobilized beads, add 60 µL from each of the 10 bead sets to the Mixing Bottle. Then add 2.4 mL Bead Diluent.

Example 2: When using 5 cytokine antibody-immobilized beads, add 60 µL from each of the 5 bead sets to the Mixing Bottle. Then add 2.7 mL Bead Diluent.

B. Preparation of Quality Controls

Before use, reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. Invert the vial several times to mix and vortex. Allow the vial to sit for 5-10 minutes. Unused portion may be stored at -20C for up to one month.

C. Preparation of Wash Buffer

Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 60 mL (2 bottles) of 10X Wash Buffer with 540 mL deionized water. Store unused portion at 2-8C for up to one month.

D. Preparation of Serum Matrix

This step is required for serum or plasma samples only.

Add 1.0 mL deionized water to the bottle containing lyophilized Serum Matrix. Mix well. Allow at least 10 minutes for complete reconstitution. Leftover reconstituted Serum Matrix should be stored at -20C for up to one month.


E. Preparation of Non-Human Primate Cytokine Panel Standard

1) Prior to use, reconstitute the Non-Human Primate Cytokine Panel Standard with 250 µL deionized water. Invert the vial several times to mix and vortex for 5-10 seconds. Allow the vial to sit for 5-10 minutes mix well. This will be Standard 7.

2) Label six polypropylene microfuge tubes Standard 6, Standard 5, Standard 4, Standard 3, Standard 2, and Standard 1, and add 150 µL of Assay Buffer to each of the six tubes. Perform 4-fold serial dilutions by adding 50 µL of the Standard 7 to the Standard 6 tube, mix well and transfer 50 µL of the Standard 6 to the Standard 5 tube, mix well and transfer 50 µL of the Standard 5 to the Standard 4 tube, mix well and transfer 50 µL of the Standard 4 to the Standard 3 tube, mix well and transfer 50 µL of the Standard 3 to the Standard 2 tube, mix well and transfer 50 µL of the Standard 2 to the Standard 1 tube, and mix Standard 1 well. The Standard 0 (Background) will be Assay Buffer.

Standard Tube #

Volume of Deionized Water to Add

Volume of Standard to Add

Standard 7 (reconstituted standard)

250 µL 0

Standard Tube #

Volume of Assay Buffer to Add

Volume of Standard to Add

Standard 6 150 µL 50 µL of Standard 7






50 µL 50 µL 50 µL 50 µL 50 µL

Standard 7

50 µL

Standard 2 Standard 1

150 µL 150 µL 150 µL 150 µL 150 µL 150 µL

Standard 3 Reconstituted Standard 7

Standard 6 Standard 5 Standard 4


After serial dilutions, the tubes should have the following concentrations for constructing standard curves.

Standard Tube #

IL-4 (pg/mL) IL-10, IL-18 (pg/mL) All Other Analytes

(pg/mL)

1 4.9 12.2 2.4

2 19.5 48.8 9.8

3 78.1 195.3 39

4 312.5 781.3 156.3

5 1,250 3,125 625

6 5,000 12,500 2,500

7 20,000 50,000 10,000


IMMUNOASSAY PROCEDURE

Prior to beginning this assay, it is imperative to read this protocol completely and to thoroughly understand the Technical Guidelines.

Allow all reagents to warm to room temperature (20-25C) before use in the assay.

Diagram the placement of Standards [0 (Background), 1, 2, 3, 4, 5, 6, 7], Controls 1 and 2, and Samples on Well Map Worksheet in a vertical configuration. (Note: Most instruments will only read the 96-well plate vertically by default.) It is recommended to run the assay in duplicate.

If using a filter plate, set the filter plate on a plate holder at all times during reagent dispensing and incubation steps so that the bottom of the plate does not touch any surface.

1. Add 200 µL of Assay Buffer into each well of the plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25C).

2. Decant Assay Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.

3. Add 25 µL of each Standard or Control into the appropriate wells. Assay Buffer should be used for Standard 0 (Background).

4. Add 25 µL of Assay Buffer to the sample wells.

5. Add 25 µL of appropriate matrix solution to the background, standards, and control wells. When assaying serum or plasma, use the Serum Matrix provided in the kit. When assaying tissue culture or other supernatant, use proper control culture medium as the matrix solution.

6. Add 25 µL of Sample into the appropriate wells.

7. Vortex Mixing Bottle and add 25 μL of the Mixed Beads to each well. (Note: During addition of Beads, shake bead bottle intermittently to avoid settling.)

8. Seal the plate with a plate sealer. Wrap the plate with foil and incubate with agitation on a plate shaker overnight (16-18 hr) at 4C or 2 hours at room temperature (20-25° C). An overnight incubation may improve assay sensitivity for some analytes.

Add 200 µL Assay Buffer per well

Add 25 µL Standard or Control to appropriate wells

Add 25 µL Assay Buffer to

background and sample wells Add 25 µL appropriate matrix

solution to background, standards, and control wells

Add 25 µL Samples to sample

wells Add 25 µL Beads to each well

Shake 10 min, RT Decant

Incubate overnight at 4°C or 2 hours at RT with shaking


9. Gently remove well contents and wash plate 2 times following instructions listed in the PLATE WASHING section.

10. Add 25 µL of Detection Antibodies into each well. (Note: Allow the Detection Antibodies to warm to room temperature prior to addition.)

11. Seal, cover with foil and incubate with agitation on a plate shaker for 1 hour at room temperature (20-25C). DO NOT ASPIRATE AFTER INCUBATION.

12. Add 25 µL Streptavidin-Phycoerythrin to each well containing the 25 µL of Detection Antibodies.

13. Seal, cover with foil and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25C).

14. Gently remove well contents and wash plate 2 times following instructions listed in the PLATE WASHING section.

15. Add 150 µL of Sheath Fluid (or Drive Fluid is using MAGPIX) to all wells. Resuspend the beads on a plate shaker for 5 minutes.

16. Run plate on Luminex 200 , HTS or FLEXMAP 3D or MAGPIX® with xPONENT software.

17. Save and analyze the Median Fluorescent Intensity (MFI) data using a 5-parameter logistic or spline curve-fitting method for calculating analyte concentrations in samples. (Note: If samples are diluted, multiply the result by the dilution factor.)

Add 25 µL Detection Antibodies per well

Incubate for 1 hour at RT Do Not Aspirate

Add 25 µL Streptavidin-Phycoerythrin per well

Incubate for 30 minutes at RT

Read on Luminex (100 µL, 50 beads per bead set)

Remove well contents and wash 2X with 200 µL Wash Buffer

Remove well contents and wash 2X with 200 µL Wash Buffer

Add 150 µL Sheath Fluid or Drive Fluid per well

PRCYTOMAG-40K Rev. 08-FEB-2011 PAGE 16 EMD MILLIPORE

PLATE WASHING 1.) Solid Plate If using a solid plate, use either a hand-held magnet or magnetic plate washer.

A.) For hand-held magnet, rest plate on magnet for 60 seconds to allow complete settling of magnetic beads. Remove well contents by gently decanting the plate in an appropriate waste receptacle and gently tapping on absorbent pads to remove residual liquid. Wash plate with 200 uL of Wash Buffer by removing plate from magnet, adding Wash Buffer, shaking for 30 seconds, reattaching to magnet, letting beads settle for 60 seconds and removing well contents as previously described after each wash. Repeat wash steps as recommended in Assay Procedure.

B.) For magnetic plate washer, let plate “soak” on magnet for 60 seconds to allow complete settling of the magnetic beads. Remove well contents by aspiration. Wash plate 2 times with 200 µL/well of Wash Buffer, letting beads “soak” for 60 seconds and removing Wash Buffer by aspiration after each wash. Repeat wash steps as recommended in Assay Procedure. Note: If using the recommended plate washer for magnetic beads (Bio-Tek ELx405) follow the appropriate equipment settings outlined on Page 14, see EQUIPMENT SETTINGS.

2.) Filter Plate (EMD Millipore Cat# MX-PLATE) If using a filter plate, use a vacuum filtration manifold to remove well contents. Wash plate 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration after each wash Repeat wash steps as recommended in the Assay Procedure. EQUIPMENT SETTINGS Bio-Tek ELx405: The general recommended wash protocol (Link Protocol) is as follows: Soak Program: Wash Program:

Soak → Aspirate→Dispense→Soak→Aspirate→Dispense→Soak→Aspirate →Dispense→Soak→Aspirate 1.) Soak program:

1. Soak duration: 60 sec 2. Shake before soak?: NO

2.) Wash program:

Method:

1. Number of cycles: 2 2. soak/shake: YES 3. Soak duration: 60 sec 4. Shake before soak: NO 5. Prime after soak: NO


Dispense:

1. Dispense volume: 200 µL/well 2. Dispense flow rate: 5 3. Dispense height: 130 (16.51 mm) 4. Horizontal disp pos: 00 (0 mm) 5. Bottom Wash first?: NO 6. Prime before start?: NO

Aspiration:

1. Aspirate height: 35 (4.445 mm) 2. Horizontal Asp Pos: 30 (1.372 mm) 3. Aspiration rate: 06 (15.0 mm/sec) 4. Aspiration delay: 0 5. Crosswise Aspir: NO 6. Final Aspir: YES 7. Final Aspir delay: 0 (0 msec)

3.) Link program: (Note: this is the program to use during actual plate washing). Link together the Soak and Wash programs outlined above. Note: After the final aspiration, there will be approximately 25 μl of residual Wash Buffer in each well. This is expected when using the BioTek Plate washer and this volume does not need to be aspirated from the plate. If using an automatic plate washer other than BioTek ELx405, please refer to the manufacturers’s recommendations for programming instructions.

These specifications are for the Luminex 200™, Luminex HTS and Luminex FLEXMAP 3D™ and MAGPIX® with xPonent software. Luminex instruments with other software (e.g. MasterPlex, StarStation, LiquiChip, Bio-Plex, LABScan100) would need to follow instrument instructions for gate settings and additional specifications from the vendors for reading Luminex Magnetic Beads. For magnetic bead assays, the Luminex 200™ and HTS instruments must be calibrated with the xPonent 3.1 compatible Calibration Kit (EMD Millipore Cat.# 40-275) and performance verified with the Performance Verification Kit (EMD Millipore Cat. # 40-276). The Luminex FLEXMap 3D™ instrument must be calibrated with the FLEXMAP 3D™ Calibrator Kit (EMD Millipore cat#40-028) and performance verified with the FLEXMAP 3D™ Performance Verification Kit (EMD Millipore cat#40-029). The Luminex MAGPIX® instrument must be calibrated with the MAGPIX® Calibration Kit (EMD Millipore cat# 40-049 and performance verified with the MAGPIX® Performance Verification Kit (EMD Millipore cat# 40-050).


NOTE: These assays cannot be run on Luminex 100™ instruments or any instruments using Luminex IS 2.3 or Luminex 1.7 software. The Luminex probe height must be adjusted to the plate provided in the kit. Please use Cat.# MAG-PLATE, if additional plates are required for this purpose.

Events: 50, per bead 50, per bead

Sample Size: 100 µL 100 µL

Gate Settings 8,000 to 15,000

Time Out 60 seconds

Bead Set: 23-Plex Premix Beads Customizable 23-Plex Beads

GM-CSF 14 GM-CSF 14

TGFα 15 TGFα 15

G-CSF 18 G-CSF 18

IFN 20 IFN 20

IL-2 33 IL-2 33

IL-10 35 IL-10 35

IL-15 37 IL-15 37

sCD40L 38 sCD40L 38

IL-17A 39 IL-17A 39

IL-1ra 42 IL-1ra 42

IL-13 44 IL-13 44

IL-1β 46 IL-1β 46

IL-4 53 IL-4 53

IL-5 55 IL-5 55

IL-6 57 IL-6 57

IL-8 63 IL-8 63

MIP-1α 65 MIP-1α 65

MCP-1 67 MCP-1 67

TNFα 72 TNFα 72

MIP-1β 73 MIP-1β 73

IL-12/23(p40) 74 IL-12/23(p40) 74

VEGF 76 VEGF 76 IL-18 78 IL-18 78

QUALITY CONTROLS

The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the EMD Millipore website www.EMD Millipore.com/techlibrary/index.do using the catalog number as the keyword.


ASSAY CHARACTERISTICS Cross-Reactivity and Cross-Species Reactivity

There was no or negligible cross-reactivity among different analytes within the panel. However, the multiplexed assay panel has been tested with samples from multiple non-human primate species.

Detection of LPS or PHA Responses In PBMC From Multiple Non-Human Primate Species1,2

Cytokine Chimpanzee Baboon Rhesus Cynomolgus Pig-Tail African Green Marmoset

IL-1ß +++ +++ +++ +++ +++ +/- +++

IL-2 +++ +++ +++ +++ +++ +++ NA

IL-4 +++ +++ +++ +++ +++ +++ -

IL-5 +++ +++ +++ +++ +++ +++ -

IL-6 +++ +++ +++ +++ +++ +++ -

TGFα NA - +++ +++ NA +++ NA

IL-8 +++ +++ +++ +++ +++ +++ -

IL-103 +++ NA +++ +++ NA NA NA

IL-12/23(p40) +++ ++ +++ ++ ++ ++ -

IL-13 +++ +++ +++ +++ +++ +++ -

IL-15 +++ +++ +++ +++ +++ +++ -

IL-17A +++ +++ +++ +++ +++ +++ +++

IL-18 +++ +++ +++ +++ +++ +++ -

IFN +++ +++ +++ +++ +++ +++ -

G-CSF +++ +++ +++ +++ +++ ++ -

GM-CSF +++ +++ +++ +++ +++ +++ -

TNFα +++ +++ +++ +++ +++ +++ -

MCP-1 +++ +++ +++ +++ +++ +++ -

IL-1ra +++ +++ +++ +++ +++ +++ +/-

sCD40L +++ +++ +++ +++ +++ +++ -

MIP-1α +++ +++ +++ +++ +++ +++ -

MIP-1ß +++ +++ +++ +++ +++ ++ +++

VEGF +++ +++ +++ +++ +++ +++ +++

1. Tested samples are from PHA/LPS stimulated PBMC from two individual animals of each species, except IL-10

2. The "+++", "++", "+/-" or "-" indicate degree of reactivity with "+++" denotes strongly reacting and "-" denotes no detectable response in LPS- or PHA-stimulated PBMC cultures. “NA” indicates data not available.

3. IL-10 Chimpanzee, Rhesus and Cynomolgus were tested as LPS challenged serum.


Assay Sensitivities (minimum detectable concentrations, pg/mL)

MinDC: Minimum Detectable Concentration is calculated by the Milliplex Analyst Immunoassay Analysis Software from EMD Millipore, It is a measure of the true limits of detection for an assay by mathematically determining what the empirical MinDC would be if an infinite number of standard concentrations were run for the assay under the same conditions.

Analyte MinDC1 (pg/mL) Average + 2SD

G-CSF 2.1 GM-CSF 1.8

IFN 1.6

IL-1 1.2

IL-1ra 2.4

IL-2 2.1

IL-4 3.1

IL-5 1.5

IL-6 1.6

IL-8 1.1

IL-10 6.4

IL-12/23(p40) 1.5

IL-13 5.8

IL-15 0.5

IL-17A 1.3

MCP-1 3.1

MIP-1β 1.6

MIP-1α 4.9

sCD40L 2.1

TGFα 1.1

TNFα 1.6

VEGF 13.6

IL-18 6.1


Precision

Intra-assay precision is generated from the mean of the %CV’s from 8 reportable results across two different concentrations of analytes in a single assay. Inter-assay precision is generated from the mean of the %CV’s across two different concentrations of analytes across 6 different assays.

Analyte Intra-Assay

(CV%) Inter-Assay

(CV%)

G-CSF < 5 < 15

GM-CSF < 5 < 15

IFN < 5 < 15

IL-1 < 5 < 15

IL-1ra < 5 < 15

IL-2 < 5 < 15

IL-4 < 5 < 15

IL-5 < 5 < 15

IL-6 < 5 < 15

IL-8 < 5 < 15

IL-10 < 5 < 15

IL-12/23(p40) < 5 < 15

IL-13 < 5 < 15

IL-15 < 5 < 15

IL-17A < 5 < 15

MCP-1 < 5 < 15

MIP-1β < 5 < 15

MIP-1α < 5 < 15

sCD40L < 5 < 15

TGFα < 5 < 15

TNFα < 5 < 15

VEGF < 5 < 15

IL-18 < 5 < 15


Accuracy

Spike Recovery: The data represent mean percent recovery of spiked standards ranging from low, medium, and high concentration in serum matrix samples (n=6).

Analyte Recovery

G-CSF 93

GM-CSF 99

IFN 92

IL-1 91

IL-1ra 90

IL-2 90

IL-4 94

IL-5 96

IL-6 87

IL-8 89

IL-10 101

IL-12/23(p40) 95

IL-13 90

IL-15 93

IL-17A 93

MCP-1 90

MIP-1β 90

MIP-1α 89

sCD40L 90

TGFα 98

TNFα 96

VEGF 70

IL-18 91


TROUBLESHOOTING GUIDE Problem Probable Cause Solution Insufficient Bead Count

Plate Washer aspirate height set too low

Adjust aspiration height according to manufacturers instructions.

Bead mix prepared inappropriately

Sonicate bead vials and vortex just prior to adding to bead mix bottle according to protocol. Agitate bead mix intermittently in reservoir while pipetting this into the plate.

Samples cause interference due to particulate matter or viscosity

See above. Also sample probe may need to be cleaned with Alcohol flush, Back flush and washes; or if needed probe should be removed and sonicated.

Probe height not adjusted correctly When reading the assay on Luminex 200™,

adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 4 alignment discs. When reading the assay on FLEXMAP 3D™, adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 1 alignment disc. When reading the assay on MAGPIX, adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 2 alignment disc.

Background is too high

Background wells were contaminated

Avoid cross-well contamination by using sealer appropriately, and pipeting with Multichannel pipets without touching reagent in plate.

Matrix used has endogenous analyte or interference

Check matrix ingredients for cross reacting components (e.g. interleukin modified tissue culture medium).

Insufficient washes Increase number of washes. Beads not in region or gate

Luminex not calibrated correctly or recently

Calibrate Luminex based on Instrument Manufacturer’s instructions, at least once a week or if temperature has changed by >3oC.

Gate Settings not adjusted correctly

Some Luminex instruments (e.g. Bioplex) require different gate settings than those described in the Kit protocol. Use Instrument default settings.

Wrong bead regions in protocol template

Check kit protocol for correct bead regions or analyte selection.

Incorrect sample type used

Samples containing organic solvents or if highly viscous should be diluted or dialyzed as required.

Instrument not washed or primed

Prime the Luminex 4 times to rid of air bubbles, wash 4 times with sheath fluid or water if there is any remnant alcohol or sanitizing liquid.


Beads were exposed to light

Keep plate and bead mix covered with dark lid or aluminum foil during all incubation steps.

Signal for whole plate is same as background

Incorrect or no Detection Antibody was added

Add appropriate Detection Antibody and continue.

Streptavidin-Phycoerythrin was not added

Add Streptavidin-Phycoerythrin according to protocol. If Detection Antibody has already been removed, sensitivity may be low.

Low signal for standard curve

Detection Antibody may have been removed prior to adding Streptavidin Phycoerythrin

May need to repeat assay if desired sensitivity not achieved.

Incubations done at inappropriate temperatures, timings or agitation

Assay conditions need to be checked.

Signals too high, standard curves are saturated

Calibration target value set too high

With some Luminex Instrument (e.g. Bio-plex) Default target setting for RP1 calibrator is set at High PMT. Use low target value for calibration and reanalyze plate.

Plate incubation was too long with standard curve and samples

Use shorter incubation time.

Sample readings are out of range

Samples contain no or below detectable levels of analyte

If below detectable levels, it may be possible to use higher sample volume. Check with tech support for appropriate protocol modifications.

Samples contain analyte concentrations higher than highest standard point.

Samples may require dilution and reanalysis for just that particular analyte.

Standard curve was saturated at higher end of curve.

See above.

High Variation in samples and/or standards

Multichannel pipet may not be calibrated

Calibrate pipets.

Plate washing was not uniform

Confirm all reagents are removed completely in all wash steps.

Samples may have high particulate matter or other interfering substances

See above.

Plate agitation was insufficient

Plate should be agitated during all incubation steps using a vertical plate shaker at a speed where beads are in constant motion without causing splashing.

Cross well contamination Check when reusing plate sealer that no reagent has touched sealer.

Care should be taken when using same pipet tips that are used for reagent additions and that pipet tip does not touch reagent in plate.

FOR FILTER PLATES ONLY Filter plate will not vacuum

Vacuum pressure is insufficient

Increase vacuum pressure such that 0.2mL buffer can be suctioned in 3-5 seconds.

Samples have insoluble Centrifuge samples just prior to assay setup


particles and use supernatant.

High lipid concentration After centrifugation, remove lipid layer and use supernatant.

Plate leaked Vacuum Pressure too high Adjust vacuum pressure such that 0.2mL buffer can be suctioned in 3-5 seconds. May need to transfer contents to a new (blocked) plate and continue.

Plate set directly on table or absorbent towels during incubations or reagent additions

Set plate on plate holder or raised edge so bottom of filter is not touching any surface.

Insufficient blotting of filter plate bottom causing wicking

Blot the bottom of the filter plate well with absorbent towels after each wash step.

Pipette touching plate filter during additions

Pipette to the side of plate.

Probe height not adjusted correctly

Adjust probe to 3 alignment discs in well H6.

Sample too viscous May need to dilute sample.


REPLACEMENT REAGENTS Cat # Non-Human Primate Cytokine Standard MXPR8040 Non-Human Primate Cytokine Quality Controls MXPR6040 Serum Matrix MXPRSM Non-human Primate Cytokine Detection Antibodies MXPR1040-2 Streptavidin-Phycoerythrin L-SAPE4 Assay Buffer L-AB Bead Diluent LBD Set of two 96-Well Plate with 2 sealers MAG-PLATE 10X Wash Buffer L-WB Antibody-Immobilized Beads

Cytokine Bead # Cat. # G-CSF 18 HGCSF-MAG GM-CSF 14 PRGMCSF-MAG IFN 20 PRIFNG-MAG IL-1β 46 HCYIL1B-MAG IL-1ra 42 HIL1RA-MAG IL-2 33 PRIL2-MAG IL-4 53 HIL4-MAG IL-5 55 HIL5-MAG IL-6 57 HCYIL6-MAG IL-8 63 HCYIL8-MAG IL-10 35 PRIL10-MAG IL-12/23(p40) 74 PRIL12P40-MAG IL-13 44 PRIL13-MAG IL-15 37 HIL15-MAG IL-17A 39 HIL17-MAG IL-18 78 PRIL18-MAG MCP-1 67 HCYMCP1-MAG MIP-1β 73 HMIP1B-MAG MIP-1α 65 PRMIP1A-MAG sCD40L 38 HCD40L-MAG TGFα 15 HCYTGFA-MAG TNFα 72 PRTNFA-MAG VEGF 76 PRVEGF-MAG Premixed 23-plex Beads PRCYPMX23-MAG


ORDERING INFORMATION

To place an order:

To assure the clarity of your custom kit order, please FAX the following information to our customer service department:

Your name, telephone and/or fax number Customer account number Shipping and billing address Purchase order number Catalog number and description of product Quantity of kits Selection of MILLIPLEX MAP Cytokine Analytes/Serum Matrix

Requirements

FAX: (636) 441-8050 Toll Free US: (800) EMD MILLIPORE

MAIL ORDERS: EMD Millipore Corp. 6 Research Park Drive St. Charles, Missouri 63304 U.S.A.

For International Customers:

To best serve our international customers in placing an order or obtaining additional information about MILLIPLEX MAP products, please contact your multiplex specialist or sales representative or email our European Customer Service at customerserviceEU@EMD Millipore.com.

Conditions of Sale

All products are for research use only. They are not intended for use in clinical diagnosis or for administration to humans or animals. All products are intended for in vitro use only.

Material Safety Data Sheets (MSDS)

Material Safety Data Sheets for EMD Millipore products may be ordered by fax or phone or through our website at www.EMD Millipore.com/techlibrary/index.do


WELL MAP

1 2 3 4 5 6 7 8 9 10 11 12

A 0 pg/mL Standard

(Background)

Standard 4

QC-1 Control

Etc.

B 0 pg/mL Standard

(Background)

Standard 4

QC-1 Control

C Standard

1 Standard

5 QC-2

Control

D Standard

1 Standard

5 QC-2

Control

E Standard

2 Standard

6 Sample

1

F Standard

2 Standard

6 Sample

1

G Standard

3

Standard

7

Sample 2

H Standard

3 Standard

7 Sample

2

Non-Human Primate Cytokine Magnetic Bead Panel 96 Well ... · Anti- IL-2 Bead 33 ˜ PRIL2-MAG ˜...

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